Monoterpene cyclases provide the focus for study of allylic pyrophosphate cyclization, a reaction type of major importance in C-C bond formation in the biosynthesis of numerous terpenoid natural products of pharmacological significance. A general stereochemical model has been proposed for the coupled isomerization-cyclization of the universal C10-isoprenoid precursor, geranyl pyrophosphate, and several key stereochemical and mechanistic elements of the scheme have been confirmed through studies on the origin of the five major bicyclic monoterpene types; pinane, bornane, fenchane, thujane and isocamphane. The noncyclizable substrate analogues, 6,7-dihydrogeranyl pyrophosphate and 2,3methanogeranyl pyrophosphate, will be employed to uncouple the isomerization step from the normally coupled reaction sequence. The respective enzymatic products, 6,7-dihydrolinalool (formed by isomerization and ionization) and homolinalyl pyrophosphate (formed by isomerization and release from the active site), will be resolved by radio-chromatographic techniques to determine the stereochemistry of the isomerization step. Specifically deuterated derivatives of [1-3H]geranyl pyrophosphate and linalyl pyrophosphate will be used to examine terminating deprotonations in monoterpene olefin formation and to determine, via kinetic isotope effects, whether pinene cyclases also synthesize stereochemically related olefins. The ability of the cyclases to transform simple monocyclic analogues of the substrate will be exploited to determine the influence of stereoelectronic features on product formation. The role of specific active site amino acid residues in substrate binding and catalysis will be explored via cyclase inhibition studies with a series of selective modifying reagents, and by determining the protective influence of the substrate and of analogues representing different substrate binding domains. A photolabile substrate analogue will be utilized for photoaffinity labeling of the presumptive hydrophobic pocket of the cyclases. Selective enzyme isolation and bulk processing methods will be combined with FPLC and a new affinity chromatography technique to obtain homogeneous cyclases with which stoichiometric relationships will be determined, and exploration of active sites initiated via the purification and sequencing of active site peptides generated by several enzyme labeling and cleavage strategies. Should this direct approach fail to yield sufficient cyclase protein, a molecular cloning and expression strategy will be adopted. The studies outlined should confirm the proposed cyclization model and its variants, provide new information on the nature of these novel catalysts, particularly the relationship of enzyme structure to reaction mechanism, and allow a clearer understanding of this important aspect of prenyl pyrophosphate metabolism.